FIG 3 .

The SufI^S12L substitution can act in cis to suppress inactivating substitutions in the SufI signal peptide twin-arginine motif. Overnight cultures of strain MC4100 ΔamiA ΔamiC ΔtatABC harboring either pTH19kr alongside pSUSufIss-mAmiA (Δtat) or pTAT101 (producing wild-type TatABC) alongside either unsubstituted pSUSufIss-mAmiA (tat⁺) or pSUSufIss-mAmiA encoding the (A) R6D, R6D-S12L, R6E, R6E-S12L, R6H, R6H-S12L, R6N, R6N-S12L, R6Q, R6Q-S12L, R5KR6K, R5KR6K-S12L, R5KR6Q, and R5KR6Q-S12L, or the (B) R5KR6H, R5KR6H-S12L, R5HR6H, and R5HR6H-S12L substitutions in the SufI signal peptide were subcultured at a 1:100 dilution, grown for a further 3 h at 37°C, pelleted, and resuspended to an OD₆₀₀ of 0.1, and 8 μl of sample was spotted onto LB agar or LB agar containing 2% SDS. Plates were incubated at 37°C for 16 h.